透過您的圖書館登入
IP:44.206.227.65
  • 學位論文

增殖性糖尿病視網膜病變患者眼內玻璃體之活性氧分析

Vitreous Levels of Reactive Oxygen Species in Proliferative Diabetic Retinopathy

指導教授 : 楊中美 楊長豪

摘要


背景說明 微血管病變是造成糖尿病病患致病與失去生命的主要原因之一,有關糖尿病視網膜病變的臨床表現、疾病分期以及有關的治療方針,都是以視網膜微血管病變的嚴重程度來作為評估的標準。因此糖尿病血管病變可說是糖尿病視網膜病變的根本來源。目前研究的結論都顯示,在糖尿病血管病變的病理生理致病過程中,氧化壓力(oxidative stress)扮演一項舉足輕重的主要角色。所謂的氧化壓力的定義是體內氧化還原狀態的不平衡,這可能是導因於體內活性氧的製造過剩,耗盡抗氧化能力(antioxidant ability),造成體內氧化力遠高於還原力。活性氧(reactive oxygen species)可以參與糖尿病血管病變的致病過程,因為活性氧本身即是一種作用在內皮細胞的第二訊息傳遞者(second messengers)。活性氧可以藉此調控血管平滑肌細胞的生長、移動與自然凋亡,內皮細胞的功能以及血管細胞外間質的形成。若是活性氧的過量產生無法受到嚴格的控制,將導致糖尿病血管病變的發生。 活性氧在眼睛發展成糖尿病視網膜病變時,扮演了一個十分重要的角色。因為視網膜是一個非常耗氧的組織,同時視網膜的組成成分中非飽和脂肪佔了一大部分,因此視網膜經由因高血糖改變的生化反應與脂肪的過氧化,容易導致活性氧與過氧化脂肪生成增加,進而引起糖尿病視網膜病變。除此之外,眼球是一個封閉的器官組織,眼內產生的氧化物質比經血漿中擴散到眼睛內更容易影響視網膜病變的嚴重程度。 目前已知血管內皮細胞生長因子(vascular endothelial growth factor; VEGF)與血管生成素-2(angiopoietin-2)在糖尿病視網膜病變致病過程中是二項非常重要的促血管新生多胜肽,兩者皆是因視網膜缺氧而刺激生成增加,也都是促進新生血管的發生。在增殖性糖尿病視網膜病變病患的玻璃體內也證明兩者皆升高。最近的研究也顯示,活性氧與血管內皮細胞生長因子和血管生成素-2有強烈的相關,可能是活性氧啟發一些特殊的訊息傳遞途徑,導致血管內皮細胞生長因子和血管生成素-2表現增加。 目的 本研究的目的是在增殖性糖尿病視網膜病變患者的玻璃體中,偵測活性氧的含量,同時分析在不同嚴重程度的糖尿病視網膜病變的患者,在玻璃體中活性氧含量是否有差異。 研究方法與病患 我們收集罹患增殖性糖尿病視網膜病變的病患,且疾病必須達到接受手術治療的適應症。這些病患在台大醫院初次接受玻璃體切除,同時願意加入本研究計畫。總計收集有三十九位患者的39隻眼睛的玻璃體樣本(實驗組),另外也收集了十六位非糖尿病患者的16隻眼睛的玻璃體樣本(控制組)進行活性氧的含量分析。所有病患的追蹤時間至少達十二個月。在增殖性糖尿病視網膜病變患者中,我們依據患者視網膜的纖維血管化增生的程度,再細分為三個組別。第一組(n=17),小於三個部位的纖維血管性局部沾黏;第二組(n=12),至少有一個大範圍沾黏或是在視神經部位、黃斑部或是大的視網膜血管上有玻璃體-視網膜沾黏;第三組(n=10),玻璃體-視網膜沾黏延伸至週邊視網膜,或是無後玻璃膜剝離,同時也可以合併有牽引性視網膜剝離;控制組(n = 16),非糖尿病患者。所有的玻璃體樣本皆是由病患同意下於常規手術中取得。所得樣本的活性氧含量分析皆是進行Luminol-enhanced的化學發光(chemiluminescence; CL)檢測。活性氧含量是以平均值 ± 標準差 CL counts/10 s來表示。結果的評估主要是三組不同嚴重程度的增殖性糖尿病視網膜病變患者與玻璃體活性氧含量之間的相關係;同時我們也研究玻璃體活性氧含量與最後手術結果是否有關聯性。最後我們利用多重因子線性迴歸分析病患的年齡、性別、糖尿病種類、患病年齡、高血壓、腎功能不全及以視網膜病變嚴重程度的分組方式等因子,來找出何種危險因子是玻璃體活性氧含量的主要決定因子。 結果 增殖性糖尿病視網膜病變患者的玻璃體活性氧含量(125.76 ± 351.72 counts/10 s)明顯的高於控制組(0.37 ± 0.72 counts/10 s; P<0.0001)。糖尿病組的玻璃體活性氧含量分別為1.86 ± 1.63 (第一組), 24.47 ± 22.68 (第二組), 與457.94 ± 597.01 counts/10s (第三組);這三組組間的差異已經達到統計上明顯的差異 (P=0.001)。迴歸分析的結果顯示只有以疾病嚴重程度的分組方式與玻璃體活性氧含量有最強且獨立性的相關(P=0.001)。最後手術的結果顯示,在第三組的患者中有三位發生無法再治療的視網膜剝離,同時這三位患者的玻璃體活性氧含量是最高的前三位。 結論 在我們的研究結果中發現增殖性糖尿病視網膜病變患者的玻璃體活性氧含量明顯高於非糖尿病患者。隨著臨床上糖尿病視網膜病變的嚴重程度加重,玻璃體活性氧含量也會隨之增加。將活性氧定量或許可以當成手術後是否成功的指標;若是指數過高,手術時可以採取更積極的治療,例如合併鞏膜扣環手術、矽油灌注或是眼內注射抗血管內皮細胞生長因子等方法。這些結果顯示活性氧可能與糖尿病視網膜病變的致病原因有相關,而且與病變的嚴重程度有正相關。將來可以延伸本研究的結果,來了解是否可以自由基清除者的藥物來降低眼內活性氧的程度,以達到預防糖尿病視網膜病變的發生,或是用以治療糖尿病視網膜病變。

並列摘要


Microangiopathies are the major cause of morbidity and mortality of diabetes mellitus (DM), contributing to diabetic retinopathy, diabetic neuropathy, and diabetic nephropathy. Oxidative stress might have an important role in the modulation of vascular smooth muscle cell growth and migration, endothelial function, and extracellular matrix formation. Many biochemical pathways are altered under hyperglycemic conditions, leading to increased generation of reactive oxygen species (ROS) and enhanced oxidative stress within tissues. In addition, total antioxidant defense capacities are reduced in plasma when oxidative stress is significantly increased. Oxidative stress might be particularly important in the development of diabetic retinopathy because the retina has a high demand for oxygen and a high unsaturated lipid content. These unique features lead to the increased production of ROS and hyperoxidative lipids through lipid peroxidation and altered biochemical reactions. Furthermore, the severity of diabetic retinopathy is more affected by locally produced oxidative substances than by those that diffuse from the serum. In the pathogenesis of diabetic retinopathy, vascular endothelial growth factor (VEGF) and angiopoietin 2 are potent angiogenic peptides that mediate ischemia-induced retinal neovascularization. VEGF and angiopoietin 2 levels are elevated in the vitreous fluid of patients with proliferative diabetic retinopathy (PDR) and might induce vascular endothelial cell proliferation in vivo. Several reports suggest that ROS are strongly correlated with increased VEGF and angiopoietin 2 expression. We hypothesized that eyes with diabetic retinopathy have a higher level of intraocular ROS than nondiabetic eyes, and that the ROS level within the vitreous reflects different degrees of severity of diabetic retinopathy. In the present study, we investigate vitreous levels of reactive oxygen species (ROS) in patients with proliferative diabetic retinopathy (PDR) and analyze ROS levels among different groups of patients with PDR. There were thirty-nine eyes of 39 patients with PDR and 16 eyes of 16 non-PDR patients (control group) who underwent primary pars plana vitrectomy for complications of PDR and other ocular diseases (control group) were enrolled, with patients’ follow-up time more than 12 months. PDR patients were classified into 3 groups according to extent of fibrovascular proliferation. Group 1 (n=17): none or focal adhesion ≤ 3 sites; Group 2 (n=12): broad adhesion ≥ 1 site(s) or vitreous-retinal adhesion around disc, macula, or arcade; Group 3 (n=10): vitreous-retinal attachment extending to periphery or no posterior vitreous detachment with or without retinal detachment; Control group (n = 16): non-PDR patients. Demographic data, clinical findings and course recorded. Vitreous samples obtained during vitrectomy and vitreous levels of ROS measured by Luminol-enhanced chemiluminescence (CL) assay. ROS levels recorded as mean ± standard deviation (SD) CL counts/10s. Correlations of vitreous levels of ROS among different groups of PDR and anatomic prognosis evaluated. Multiple linear regression analysis of selective potential risk factors performed to investigate the main determinants of ROS levels. Vitreous level of ROS was significantly higher in patients with PDR (125.76 ± 351.72 CL counts/10s) than in control patients (0.37 ± 0.72 CL counts/10s) (P<0.0001). ROS levels (CL counts/10s) in Groups 1, 2, and 3 were 1.86 ± 1.63, 24.47 ± 22.68, and 457.94 ± 597.01, respectively; the difference among groups was significant (P=0.001). Regression analysis showed only patient grouping (according to severity of fibrovascular proliferation) manifested strongest dependent association with ROS levels (P=0.001). Final anatomic results revealed that recurrent untreatable retinal detachment occurred in 3 patients of Group 3, who also had the highest ROS levels (1784.40; 1076.22 and 896.29 CL counts/10s). In conclusion, our results suggest that ROS levels are significantly elevated in the vitreous fluid of PDR patients, and that ROS levels correlate with the severity of PDR. Quantification of the vitreous levels of ROS might be an indicator of the anatomic results and more aggressive treatments, such as combined scleral buckling, silicone oil infusion, or intravitreal anti-VEGF agent injection should be undertaken during surgery. These findings suggest an important association of ROS and the pathogenesis of diabetic retinopathy. Further extended studies are needed to elucidate whether the reduction of ROS by free radical scavenger therapies might be a new approach for inhibiting the development of diabetic retinopathy.

參考文獻


Altomare E, Grattagliano I, Vendemaile G, et al. Oxidative protein damage in human diabetic eye: evidence of a retinal participation. Eur J Clin Invest 1997; 27:141-7.
Baudouin C, Ettaiche M, Imbert F, et al. Inhibition of preretinal proliferation by free radical scavengers in an experimental model of tractional retinal detachment. Exp Eye Res 1994; 59:697-706.
Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001;414:813-20.
Cai H. NAD(P)H oxidase-dependent self-propagation of hydrogen peroxide and vascular disease. Circ Res 2005;96: 818–22.
Chien CT, Chang WT, Chen HW, et al. Ascorbate supplement reduces oxidative stress in dyslipidemic patients undergoing apheresis. Arterioscler Thromb Vasc Biol 2004; 24:1111-7.

延伸閱讀